Method of isolating human neuroepithelial precursor cells from human fetal tissue

a technology of human neuroepithelial precursor cells and human fetal tissue, which is applied in the direction of artificial cell constructs, biocide, drug compositions, etc., can solve the problems of limited availability of cross-reactive reagents in the comparison study of rodent and human stem cells

Inactive Publication Date: 2005-06-09
UNIV OF UTAH RES FOUND
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  • Summary
  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

Indeed, comparative studies of rodent and human-derived stem cells have been hampered by the limited availability of cross-reactive reagents.

Method used

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Examples

Experimental program
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Effect test

example 1

Culture of Human Neural Stem Cells

[0038] Human neural progenitor cells derived from fetal tissue were acquired from Clonetics (San Diego, Calif.). Frozen aliquots of cells were thawed and plated on fibronectin / laminin-coated multiwell dishes in Neural Progenitor Cell Basal Medium (NPBM, Clonetics) supplemented with human recombinant basic fibroblast growth factor, human recombinant epidermal growth factor, neural survival factors, 5 mg / ml gentamicin and 5 μg / ml amphotericin-B (Singlequots, Clonetics). Cultures were incubated at 37° C., 5% CO2 and fixed 24 hours later. These wells were subsequently processed for immunocytochemistry to assess the starting population of Clonetics cells. In parallel, Clonetics cells were thawed and immediately plated on fibronectin / laminin-coated flasks (Greiner) and cultured in Neuroepithelial Precursor (NEP) medium that consisted of DMEM-F12 (Life Technologies, Grand Island, N.Y.) supplemented with additives as described by Bottenstein and Sate (Pro...

example 2

Isolation of Human Neuroepithelial Precursor Cells (hNEPs)

[0039] After 5 days in culture, immunopanning and flow-activated cell sorting were used to remove eNCAM+, NG2+, and A2B5+ cells. Briefly, cells were treated with 5 mM EDTA (Life Technologies) and the suspension plated on an eNCAM antibody (5A5, Developmental Studies Hybridoma Bank)-coated dish to allow binding of all eNCAM+ cells to the plate. ENCAM antibody-coated dishes were prepared by sequentially coating tissue culture dishes with an unlabeled anti-mouse IgM antibody (10 μg / ml) overnight, rinsing dishes with DPBS, followed by coating with 5A5 hybridoma supernatant for 1-3 hours at 37° C. Plates were washed twice with DPBS prior to plating neural progenitor cells. After a 30 minute exposure period, unbound cells (eNCAM cells) were removed and plated onto a dish coated with antibodies to NG2 for 30 minutes. NG2 panning dishes were made by coating dishes with an NG2 antibody (1:50) for 1-3 hours at 37° C. The supernatant ...

example 3

Generation of Neurons, Oligodendrocytes and Astrocytes from hNEPs

[0040] Panned / sorted populations of human NEPs were plated on fibronectin / laminin-coated 12 mm coverslips in various conditions to promote differentiation. Coverslips were fixed with 4% paraformaldehyde at the times established below. To induce neuronal differentiation, cells were exposed to bFGF 10 ng / ml) and NT3 (10 ng / ml, Peprotech). After 5 days in culture, fixed cultures were stained using antibodies to β-III tubulin to assess the capacity of these cells to differentiate into neurons. For oligodendrocyte differentiation, cells were plated in a bFGF (10 ng / ml)-containing medium for 2 days and then were switched to a medium containing PDGF (10 mg / ml, Upstate Biotech, Waltham, Mass.) and T3 (50 nM, Sigma Chemical Co. St. Louis, Mo.) for 7 days. Antibodies to O4 were used to identify oligodendrocytes in culture. For astrocytic differentiation, cells were cultured for 5 days in the presence of fetal calf serum (10%, ...

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Abstract

A method for isolating human neuroepithelial precursor cells from human fetal tissue by culturing the human fetal cells in fibroblast growth factor and chick embryo extract and immunodepleting from the cultured human fetal cells any cells expressing A2B5, NG2 and eNCAM is provided. In addition, methods for transplanting these cells into an animal are provided. Animals models transplanted with these human neuroepithelial precursor cells and methods for monitoring survival, proliferation, differentiation and migration of the cells in the animal model via detection of human specific markers are also provided.

Description

BACKGROUND OF THE INVENTION [0001] The demonstration that stem cells exist in the adult brain and spinal cord (Chiasson et al. J. Neurosci. 1999 19:4462-71; Doetsch et al. Cell 1999 97:703-16; Gage et al. J. Neurobiol. 1998 36:249-66; Johansson et al. Exp. Cell Res. 1999 253: 733-6; Kukekov et al. Exp. Neurol. 1999 156:333-44; Pagano et al. Stem Cells 2000 18:295-300; Palmer et al. J. Neurosci. 1999 19:8487-97; Weiss et al. J. Neurosci. 1996 16:7599-609), that neurogenesis and gliogenesis are ongoing processes (Eriksson et al. Nat. Med. 1998 4:1313-7; Horner et al. J. Neurosci. 2000 20:2218-28; Johansson et al. Cell 1999 96:25-34; Kirschenbaum et al. Cereb. Cortex 1994 4:576-89) and that stem cell populations can be modulated by extrinsic signals (Ahmed et al. J. Neurosci. 1995 15:5765-78; Forsberg-Nilsson et al. J. Neurosci. Res. 1998 53:521-30; Johe et al. Genes Dev. 1996 10:3219-40; Kalyani et al. Dev. Biol. 1997 186:202-23; Palmer et al. J. Neurosci. 1999 19:8487-97; Tsai, R. Y....

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K35/12C12N5/0797
CPCA61K35/12A61K35/54C12N5/0623A61P25/28
Inventor MAYER-PROSCHEL, MARGOTRAO, MAHENDRA S.TRESCO, PATRICK A.MESSINA, DARIN J.
Owner UNIV OF UTAH RES FOUND
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